title: Heterogeneity in the molecular composition of excitatory postsynaptic sites during development of hippocampal neurons in culture.
abstract: To determine their roles in the assembly of glutamatergic postsynaptic sites, we studied the distributions of NMDA- and AMPA-type glutamate receptors; the NMDA receptor-interacting proteins alpha-actinin-2, PSD-95, and chapsyn; and the PSD-95-associated protein GKAP during the development of hippocampal neurons in culture. NMDA receptors first formed nonsynaptic proximal dendrite shaft clusters within 2-5 d. AMPA receptors were diffuse at this stage and began to cluster on spines at 9-10 d. NMDA receptor clusters remained partially nonsynaptic and mainly distinct from AMPA receptor clusters until after 3 weeks in culture, when the two began to colocalize at spiny synaptic sites. Thus, the localization of NMDA and AMPA receptors must be regulated by different mechanisms. alpha-Actinin-2 colocalized with the NMDA receptor only at spiny synaptic clusters, but not at shaft nonsynaptic or synaptic clusters, suggesting a modulatory role in the anchoring of NMDA receptor at spines. PSD-95, chapsyn, and GKAP were present at some, but not all, nonsynaptic NMDA receptor clusters during the first 2 weeks, indicating that none is essential for NMDA receptor cluster formation. When NMDA receptor clusters became synaptic, PSD-95 and GKAP were always present, consistent with an essential function in synaptic localization of NMDA receptors. Furthermore, PSD-95 and GKAP clustered opposite presynaptic terminals several days before either NMDA or AMPA receptors clustered at these presumptive postsynaptic sites. These results suggest that synapse development proceeds by formation of a postsynaptic scaffold containing PSD-95 and GKAP in concert with presynaptic vesicle clustering, followed by regulated attachment of glutamate receptor subtypes to this scaffold.
authors.affiliation: Department of Cell and Structural Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.
authors.forename: A
authors.initials: A
authors.lastname: Rao
authors.forename: E
authors.initials: E
authors.lastname: Kim
authors.forename: M
authors.initials: M
authors.lastname: Sheng
authors.forename: A M
authors.initials: AM
authors.lastname: Craig
mesh_terms: D000185:Actinin / Q000378:metabolism; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D002478:Cells, Cultured; D016922:Cellular Senescence / Q000502:physiology; D000076122:Disks Large Homolog 4 Protein; D004622:Embryo, Mammalian / Q000166:cytology / Q000502:physiology; D005314:Embryonic and Fetal Development / Q000502:physiology; D006624:Hippocampus / Q000166:cytology / Q000196:embryology*; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins / Q000502:physiology; D009433:Neural Inhibition / Q000502:physiology; D009474:Neurons / Q000502:physiology*; D051381:Rats / Q000196:embryology; D011940:Receptor Aggregation / Q000502:physiology; D018091:Receptors, AMPA / Q000378:metabolism / Q000502:physiology; D016194:Receptors, N-Methyl-D-Aspartate / Q000378:metabolism / Q000502:physiology; D000074407:SAP90-PSD95 Associated Proteins; D013569:Synapses / Q000502:physiology*
chemical_list: D002352:Carrier Proteins; D000076122:Disks Large Homolog 4 Protein; C501449:Dlg4 protein, rat; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D018091:Receptors, AMPA; D016194:Receptors, N-Methyl-D-Aspartate; D000074407:SAP90-PSD95 Associated Proteins; C033652:postsynaptic density proteins; D000185:Actinin